PROJECT SUMMARY/ABSTRACT Hedgehog (Hh) proteins are secreted morphogens that regulate normal cell differentiation as well as malignant cell growth. Covalent attachment of the fatty acid palmitate to the N-terminus of Hh is critical for Hh function. Unlike nearly all other palmitoylated proteins, that contain thioester linked palmitate, palmitate is linked to Hh via amide (N-) bond. The overall goals of this research are to elucidate the enzymology and biochemical mechanism of N-palmitoylation of developmentally important signaling proteins, and to understand how expression and function of N-palmitoyl transferases is regulated. We will use Hh proteins as model systems to address the following issues: 1. To reconstitute N-palmitoylation using purified Hedgehog and Rasp proteins Genetic experiments in flies and mice indicate that Rasp, a multipass membrane protein, is required for Hh palmitoylation. To date, there is no biochemical evidence that Rasp, or its mammalian homolog Mart-2, functions independently and catalytically as a palmitoyl transferase. We have now succeeded in purifying Mart -2 to homogeneity in active form. The biochemical parameters and enzymatic mechanism of Shh and Hh N-palmitoylation will be determined. 2. Structure/Function analysis of Rasp/Mart-2 and Hh/Shh: How do MBOAT proteins work and how do they recognize their substrates? Using deletion analysis and chimeric proteins formed between Rasp and another MBOAT family member Porcupine, we will identify the transmembrane and/or cytoplasmic loop regions of Rasp and Mart-2 that comprise the active site and are important for catalysis. We will identify the minimum N-palmitoylation sequence motif within Hh/Shh and use this information to identify other substrates for Rasp and Mart-2. 3. To determine how N-Palmitoylation by Rasp/Mart-2 is regulated within the cell Subcellular localization and trafficking experiments will be performed to determine when and where Hh is palmitoylated. Methods to inhibit Mart-2 mediated palmitoylation will be devised. A high throughput screen will be exploited to identify novel small molecular inhibitors of Hh palmitoylation. These reagents could potentially be clinically useful as anti-tumor agents in Hh driven malignancies. The effects of Mart-2 inhibition on growth of Shh-dependent pancreatic cancer cells will be assayed. Project Narrative/Relevance to Public Health: Hh signaling has been shown to drive the growth of many human cancers, including medulloblastoma, melanoma, and pancreatic tumors. The proposed studies will help us understand how Hh proteins work in normal and malignant cells and will aim to develop Hh inhibitors that could potentially be clinically useful as anti-tumor agents in Hh driven malignancies.